The Selenoproteome as a Dynamic Response Mechanism to Oxidative Stress in Hydrogenotrophic Methanogenic Communities.

Methanogenesis is a critical process in the carbon cycle that is applied industrially in anaerobic digestion and biogas production. While naturally occurring in diverse environments, methanogenesis requires anaerobic and reduced conditions, although varying degrees of oxygen tolerance have been described. Microaeration is suggested as the next step to increase methane production and improve hydrolysis in digestion processes; therefore, a deeper understanding of the methanogenic response to oxygen stress is needed. To explore the drivers of oxygen tolerance in methanogenesis, two parallel enrichments were performed under the addition of H2/CO2 in an environment without reducing agents and in a redox-buffered environment by adding redox mediator 9,10-anthraquinone-2,7-disulfonate disodium. The cellular response to oxidative conditions is mapped using proteomic analysis. The resulting community showed remarkable tolerance to high-redox environments and was unperturbed in its methane production. Next to the expression of pathways to mitigate reactive oxygen species, the higher redox potential environment showed an increased presence of selenocysteine and selenium-associated pathways. By including sulfur-to-selenium mass shifts in a proteomic database search, we provide the first evidence of the dynamic and large-scale incorporation of selenocysteine as a response to oxidative stress in hydrogenotrophic methanogenesis and the presence of a dynamic selenoproteome.

Study on the accelerated biodegradation of PAHs in subsurface soil via coupled low-temperature thermally treatment and electron acceptor stimulation based on metagenomic sequencing.

In situ anoxic bioremediation is a sustainable technology to remediate PAHs contaminated soils. However, the limited degradation rate of PAHs under anoxic conditions has become the primary bottleneck hindering the application of this technology. In this study, coupled low-temperature thermally treatment (<50 °C) and EA biostimulation was used to enhance PAH removal. Anoxic biodegradation of PAHs in soil was explored in microcosms in the absence and presence of added EAs at 3 temperatures (15 °C, 30 °C, and 45 °C). The influence of temperature, EA, and their interaction on the removal of PAHs were identified. A PAH degradation model based on PLSR analysis identified the importance and the positive/negative role of parameters on PAH removal. Soil archaeal and bacterial communities showed similar succession patterns, the impact of temperature was greater than that of EA. Soil microbial community and function were more influenced by temperature than EAs. Close and frequent interactions were observed among soil bacteria, archaea, PAH-degrading genes and methanogenic genes. A total of 15 bacterial OTUs, 1 PAH-degrading gene and 2 methanogenic genes were identified as keystones in the network. Coupled low-temperature thermally treatment and EA stimulation resulted in higher PAH removal efficiencies than EA stimulation alone and low-temperature thermally treatment alone.

Distribution of extracellular amino acids and their potential functions in microbial cross-feeding in anaerobic digestion systems.

Anaerobic digestion is a prevalent bioenergy production process relying on a complex network of symbiotic interactions, where the nutrient based cross-feeding is an essential microbial mechanism. Here, the cross-feeding function was assessed by analyzing extracellular polymeric substances-associated amino acids in microbial aggregates collected from 14 lab-scale anaerobic digesters, as well as deciphering their genetically biosynthetic potential by syntrophic bacteria and methanogens. The total concentration of essential amino acids ranged from 1.2 mg/g VSS to 174.0 mg/g VSS. The percentages of glutamic acid (8.5 âˆ¼ 37.6%), lysine (2.7 âˆ¼ 22.6%), alanine (5.6 âˆ¼ 13.2%), and valine (3.0 âˆ¼ 10.4%) to the total amount of detected amino acids were the highest in most samples. Through metagenomics analysis, several investigated syntrophs (i.e., Smithella, Syntrophobacter, Syntrophomonas, and Mesotoga) and methanogens (i.e., Methanothrix and Methanosarcina) were auxotrophies, but the genetic ability of syntrophs and methanogens to synthesize some essential amino acids could be complementary, implying potential cross-feeding partnership.

Anti-Methanogenic Traits of Safflower Oil Compounds Against Methyl-Coenzyme M Reductase Receptor in Equines: An In Silico Docking Analysis.

Greenhouse gases emission from livestock is the major concern for the ecosystem. Despite the lower contribution of non-ruminants towards greenhouse gas emission as compared to the ruminants, the emission of methane (CH4) gas from equines is expected to be increased in future due to its increasing population. Thus, it is essential to find or screen potential anti-methanogenic agent in a cost-effective and quicker manner. Considering this, the present investigation was aimed to analyze anti-methanogenic characteristic of bioactive compounds of safflower oil by targeting methanogenesis catalyzing enzyme (Methyl-coenzyme M reductase; MCR) via in silico tool. Initially, a total of 25 compounds associated with safflower oil were selected and their drug-likeness traits were predicted through Lipinski's rule of 5. Of 25 compounds, 9 compounds passed all the parameters of Lipinski's rule of five. These 9 ligands were further submitted for ADME traits analysis using Swiss ADME tool. Results revealed the absence of Lipinski's violation and approval of drug-likeness attributes of methyl tetradecanoate, 3-isopropyl-6-methylenecyclohex-1-ene, trans-2,4-decadienal, cis-6-nonenal, limonene, syringic acids, matairesinol, acacetin, and 2,5-octanedione. Molecular docking analysis was performed for analyzing the affinity between the selected 9 ligands and MCR receptor using FRED v3.2.0 from OpenEye Scientific Software and Discovery Studio client v16.1.0. Results showed maximum binding interaction of acacetin with MCR with the chemguass4 score of -13.35. Other ligands showed comparatively lower binding affinity in the order of matairesinol (-12.43) > methyl tetradecanoate (-9.25) > cis-6-nonenal (-7.88) > syringic acids (-7.73) > limonene (-7.18) > trans-2,4-decadienal (-7.07) > 3-isopropyl-6-methylenecyclohex-1-ene (-7.01) > 2,5-octanedione (-7.0.). In a nutshell, these identified compounds were observed as potential agents to reduce CH4 production from equines by targeting MCR. This in silico study emphasized the role of safflower-associated compounds in developing anti-methanogenic drug for equines in future.

Several ways one goal-methanogenesis from unconventional substrates.

Methane is the second most important greenhouse gas on earth. It is produced by methanogenic archaea, which play an important role in the global carbon cycle. Three main methanogenesis pathways are known: in the hydrogenotrophic pathway H2 and carbon dioxide are used for methane production, whereas in the methylotrophic pathway small methylated carbon compounds like methanol and methylated amines are used. In the aceticlastic pathway, acetate is disproportionated to methane and carbon dioxide. However, next to these conventional substrates, further methanogenic substrates and pathways have been discovered. Several phylogenetically distinct methanogenic lineages (Methanosphaera, Methanimicrococcus, Methanomassiliicoccus, Methanonatronarchaeum) have evolved hydrogen-dependent methylotrophic methanogenesis without the ability to perform either hydrogenotrophic or methylotrophic methanogenesis. Genome analysis of the deep branching Methanonatronarchaeum revealed an interesting membrane-bound hydrogenase complex affiliated with the hardly described class 4 g of multisubunit hydrogenases possibly providing reducing equivalents for anabolism. Furthermore, methylated sulfur compounds such as methanethiol, dimethyl sulfide, and methylmercaptopropionate were described to be converted into adapted methylotrophic methanogenesis pathways of Methanosarcinales strains. Moreover, recently it has been shown that the methanogen Methermicoccus shengliensis can use methoxylated aromatic compounds in methanogenesis. Also, tertiary amines like choline (N,N,N-trimethylethanolamine) or betaine (N,N,N-trimethylglycine) have been described as substrates for methane production in Methanococcoides and Methanolobus strains. This review article will provide in-depth information on genome-guided metabolic reconstructions, physiology, and biochemistry of these unusual methanogenesis pathways. KEY POINTS: • Newly discovered methanogenic substrates and pathways are reviewed for the first time. • The review provides an in-depth analysis of unusual methanogenesis pathways. • The hydrogenase complex of the deep branching Methanonatronarchaeum is analyzed.

Metabolic versatility of freshwater sedimentary archaea feeding on different organic carbon sources.

Members of the phylum Bathyarchaeota and the class Thermoplasmata are widespread in marine and freshwater sediments where they have been recognized as key players in the carbon cycle. Here, we tested the responsiveness of archaeal communities on settled plant debris and sediment from a karstic lake to different organic carbon amendments (amino acids, plant-derived carbohydrates, and aromatics) using a lab-scale microcosm. Changes in the composition and abundance of sediment and biofilm archaeal communities in both DNA and RNA fractions were assessed by 16S rRNA gene amplicon sequencing and qPCR, respectively, after 7 and 30 days of incubation. Archaeal communities showed compositional changes in terms of alpha and beta diversity in relation to the type of carbon source (amino acids vs. plant-derived compounds), the nucleic acid fraction (DNA vs. RNA), and the incubation time (7 vs. 30 days). Distinct groups within the Bathyarchaeota (Bathy-15 and Bathy-6) and the Thermoplasmata (MBG-D) differently reacted to carbon supplements as deduced from the analysis of RNA libraries. Whereas Bathyarchaeota in biofilms showed a long-term positive response to humic acids, their counterparts in the sediment were mainly stimulated by the addition of tryptophan, suggesting the presence of different subpopulations in both habitats. Overall, our work presents an in vitro assessment of the versatility of archaea inhabiting freshwater sediments towards organic carbon and introduces settled leaf litter as a new habitat for the Bathyarchaeota and the Thermoplasmata.

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs.

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 µmol CH4 g-1 oil d-1 , orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC-MS and FTICR-MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.

Conductive Iron Oxides Promote Methanogenic Acetate Degradation by Microbial Communities in a High-Temperature Petroleum Reservoir.

Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by microbial communities enriched from the production water of a high-temperature petroleum reservoir. A microbial community analysis revealed that Petrothermobacter spp. (phylum Deferribacteres), known as thermophilic Fe(III) reducers, predominated in the magnetite-supplemented enrichment, whereas other types of Fe(III) reducers, such as Thermincola spp. and Thermotoga spp., were dominant under ferrihydrite-reducing conditions. These results suggest that magnetite induced interspecies electron transfer via electric currents through conductive particles between Petrothermobacter spp. and methanogens. This is the first evidence for possible electric syntrophy in high-temperature subsurface environments.

Nitrate decreases ruminal methane production with slight changes to ruminal methanogen composition of nitrate-adapted steers.

BACKGROUND: This study was conducted to examine effects of nitrate on ruminal methane production, methanogen abundance, and composition. Six rumen-fistulated Limousin×Jinnan steers were fed diets supplemented with either 0% (0NR), 1% (1NR), or 2% (2NR) nitrate (dry matter basis) regimens in succession. Rumen fluid was taken after two-week adaptation for evaluation of in vitro methane production, methanogen abundance, and composition measurements. RESULTS: Results showed that nitrate significantly decreased in vitro ruminal methane production at 6 h, 12 h, and 24 h (P < 0.01; P < 0.01; P = 0.01). The 1NR and 2NR regimens numerically reduced the methanogen population by 4.47% and 25.82% respectively. However, there was no significant difference observed between treatments. The alpha and beta diversity of the methanogen community was not significantly changed by nitrate either. However, the relative abundance of the methanogen genera was greatly changed. Methanosphaera (PL = 0.0033) and Methanimicrococcus (PL = 0.0113) abundance increased linearly commensurate with increasing nitration levels, while Methanoplanus abundance was significantly decreased (PL = 0.0013). The population of Methanoculleus, the least frequently identified genus in this study, exhibited quadratic growth from 0% to 2% when nitrate was added (PQ = 0.0140). CONCLUSIONS: Correlation analysis found that methane reduction was significantly related to Methanobrevibacter and Methanoplanus abundance, and negatively correlated with Methanosphaera and Methanimicrococcus abundance.

Thermodynamically diverse syntrophic aromatic compound catabolism.

Specialized organotrophic Bacteria 'syntrophs' and methanogenic Archaea 'methanogens' form a unique metabolic interaction to accomplish cooperative mineralization of organic compounds to CH4 and CO2 . Due to challenges in cultivation of syntrophs, mechanisms for how their organotrophic catabolism circumvents thermodynamic restrictions remain unclear. In this study, we investigate two communities hosting diverse syntrophic aromatic compound metabolizers (Syntrophus, Syntrophorhabdus, Pelotomaculum and an uncultivated Syntrophorhabdacaeae member) to uncover their catabolic diversity and flexibility. Although syntrophs have been generally presumed to metabolize aromatic compounds to acetate, CO2 , H2 and formate, combined metagenomics and metatranscriptomics show that uncultured syntrophs utilize unconventional alternative metabolic pathways in situ producing butyrate, cyclohexanecarboxylate and benzoate as catabolic byproducts. In addition, we also find parallel utilization of diverse H2 and formate generating pathways to facilitate interactions with partner methanogens. Based on thermodynamic calculations, these pathways may enable syntrophs to combat thermodynamic restrictions. In addition, when fed with specific substrates (i.e., benzoate, terephthalate or trimellitate), each syntroph population expresses different pathways, suggesting ecological diversification among syntrophs. These findings suggest we may be drastically underestimating the biochemical capabilities, strategies and diversity of syntrophic bacteria thriving at the thermodynamic limit.

Iron oxides alter methanogenic pathways of acetate in production water of high-temperature petroleum reservoir.

Acetate is a key intermediate in anaerobic crude oil biodegradation and also a precursor for methanogenesis in petroleum reservoirs. The impact of iron oxides, viz. ß-FeOOH (akaganéite) and magnetite (Fe3O4), on the methanogenic acetate metabolism in production water of a high-temperature petroleum reservoir was investigated. Methane production was observed in all the treatments amended with acetate. In the microcosms amended with acetate solely about 30% of the acetate utilized was converted to methane, whereas methane production was stimulated in the presence of magnetite (Fe3O4) resulting in a 48.34% conversion to methane. Methane production in acetate-amended, ß-FeOOH (akaganéite)-supplemented microcosms was much faster and acetate consumption was greatly improved compared to the other conditions in which the stoichiometric expected amounts of methane were not produced. Microbial community analysis showed that Thermacetogenium spp. (known syntrophic acetate oxidizers) and hydrogenotrophic methanogens closely related to Methanothermobacter spp. were enriched in acetate and acetate/magnetite (Fe3O4) microcosms suggesting that methanogenic acetate metabolism was through hydrogenotrophic methanogenesis fueled by syntrophic acetate oxidizers. The acetate/ß-FeOOH (akaganéite) microcosms, however, differed by the dominance of archaea closely related to the acetoclastic Methanosaeta thermophila. These observations suggest that supplementation of ß-FeOOH (akaganéite) accelerated the production of methane further, driven the alteration of the methanogenic community, and changed the pathway of acetate methanogenesis from hydrogenotrophic methanogenesis fueled by syntrophic acetate oxidizers to acetoclastic.

Activation of CO2-reducing methanogens in oil reservoir after addition of nutrient.

Nutrient addition as part of microbial enhanced oil recovery (MEOR) operations have important implications for more energy recovery from oil reservoirs, but very little is known about the in situ response of microorganisms after intervention. An analysis of two genes as biomarkers, mcrA encoding the key enzyme in methanogenesis and fthfs encoding the key enzyme in acetogenesis, was conducted during nutrient addition in oil reservoir. Clone library data showed that dominant mcrA sequences changed from acetoclastic (Methanosaetaceae) to CO2-reducing methanogens (Methanomicrobiales and Methanobacteriales), and the authentic acetogens affiliated to Firmicutes decreased after the intervention. Principal coordinates analysis (PCoA) and Jackknife environment clusters revealed evidence on the shift of the microbial community structure among the samples. Quantitative analysis of methanogens via qPCR showed that Methanobacteriales and Methanomicrobiales increased after nutrient addition, while acetoclastic methanogens (Methanosaetaceae) changed slightly. Nutrient treatment activated native CO2-reducing methanogens in oil reservoir. The high frequency of Methanobacteriales and Methanomicrobiales (CO2-reducers) after nutrient addition in this petroleum system suggested that CO2-reducing methanogenesis was involved in methane production. The nutrient addition could promote the methane production. The results will likely improve strategies of utilizing microorganisms in subsurface environments.

Bioactive fractions from the pasture legume Biserrula pelecinus L. have an anti-methanogenic effect against key rumen methanogens.

Methanogenic archaea (methanogens) are common inhabitants of the mammalian intestinal tract. In ruminants, they are responsible for producing abundant amounts of methane during digestion of food, but selected bioactive plants and compounds may inhibit this activity. Recently, we have identified that, Biserrula pelecinus L. (biserrula) is one such plant and the current study investigated the specific anti-methanogenic activity of the plant. Bioassay-guided extraction and fractionation, coupled with in vitro fermentation batch culture were used to select the most bioactive fractions of biserrula. The four fractions were then tested against five species of methanogens grown in pure culture. Fraction bioactivity was assessed by measuring methane production and amplification of the methanogen mcrA gene. Treatments that showed bioactivity were subcultured in fresh broth without the bioactive fraction to distinguish between static and cidal effects. All four fractions were active against pure cultures, but the F2 fraction was the most consistent inhibitor of both methane production and cell growth, affecting four species of methanogens and also producing equivocal-cidal effects on the methanogens. Other fractions had selective activity affecting only some methanogens, or reducing either methane production or methanogenic cell growth. In conclusion, the anti-methanogenic activity of biserrula can be linked to compounds contained in selected bioactive fractions, with the F2 fraction strongly affecting key rumen methanogens. Further study is required to identify the specific plant compounds in biserrula that are responsible for the anti-methanogenic activity. These findings will help devise novel strategies to control methanogen populations and activity in the rumen, and consequently contribute in reducing greenhouse gas emissions from ruminants.

Effects and mode of action of chitosan and ivy fruit saponins on the microbiome, fermentation and methanogenesis in the rumen simulation technique.

This study investigates the effects of supplementing a control diet (CON) with chitosan (CHI) or ivy fruit saponins (IVY) as natural feed additives. Both additives had similar abilities to decrease rumen methanogenesis (-42% and -40%, respectively) using different mechanisms: due to its antimicrobial and nutritional properties CHI promoted a shift in the fermentation pattern towards propionate production which explained about two thirds of the decrease in methanogenesis. This shift was achieved by a simplification of the structure in the bacterial community and a substitution of fibrolytic (Firmicutes and Fibrobacteres) by amylolytic bacteria (Bacteroidetes and Proteobacteria) which led to greater amylase activity, lactate and microbial protein yield with no detrimental effect on feed digestibility. Contrarily, IVY had negligible nutritional properties promoting minor changes in the fermentation pattern and on the bacterial community. Instead, IVY modified the structure of the methanogen community and decreased its diversity. This specific antimicrobial effect of IVY against methanogens was considered its main antimethanogenic mechanism. IVY had however a negative impact on microbial protein synthesis. Therefore, CHI and IVY should be further investigated in vivo to determine the optimum doses which maintain low methanogenesis but prevent negative effects on the rumen fermentation and animal metabolism.

Biodegradation of C7 and C8 iso-alkanes under methanogenic conditions.

Iso-alkanes comprise a substantial proportion of petroleum and refined products that impact the environment, but their fate is cryptic under methanogenic conditions. We investigated methanogenic biodegradation of C7 and C8 iso-alkanes found in naphtha, specifically 2-methylhexane, 3-methylhexane, 2-methylheptane, 4-methylheptane and 3-ethylhexane. These were incubated as a mixture or individually with enrichment cultures derived from oil sands tailings ponds that generate methane from naphtha components; substrate depletion and methane production were monitored for up to 663 days. 3-Methylhexane and 4-methylheptane were degraded both singly and in the mixture, whereas 2-methylhexane and 2-methylheptane resisted degradation as single substrates but were depleted in the iso-alkane mixture, suggesting co-metabolism. 3-Ethylhexane was degraded neither singly nor with co-substrates. Putative metabolites consistent with succinylated C7 and C8 were detected, suggesting activation by addition of iso-alkanes to fumarate and corresponding to detection of alkylsuccinate synthase-like genes. 454 pyrotag sequencing, cloning and terminal restriction fragment length polymorphism of 16S rRNA genes revealed predominance of a novel member of the family Peptococcaceae (order Clostridiales) and Archaea affiliated with Methanoregula and Methanosaeta. We report here isomer-specific metabolism of C7 -C8 iso-alkanes under methanogenic conditions and propose their activation by a novel Peptococcaceae via addition to fumarate.

Gas formation. Formation temperatures of thermogenic and biogenic methane.

Methane is an important greenhouse gas and energy resource generated dominantly by methanogens at low temperatures and through the breakdown of organic molecules at high temperatures. However, methane-formation temperatures in nature are often poorly constrained. We measured formation temperatures of thermogenic and biogenic methane using a "clumped isotope" technique. Thermogenic gases yield formation temperatures between 157° and 221°C, within the nominal gas window, and biogenic gases yield formation temperatures consistent with their comparatively lower-temperature formational environments (<50°C). In systems where gases have migrated and other proxies for gas-generation temperature yield ambiguous results, methane clumped-isotope temperatures distinguish among and allow for independent tests of possible gas-formation models.

Deep subsurface sulfate reduction and methanogenesis in the Iberian Pyrite Belt revealed through geochemistry and molecular biomarkers.

The Iberian Pyrite Belt (IPB, southwest of Spain), the largest known massive sulfide deposit, fuels a rich chemolithotrophic microbial community in the Río Tinto area. However, the geomicrobiology of its deep subsurface is still unexplored. Herein, we report on the geochemistry and prokaryotic diversity in the subsurface (down to a depth of 166 m) of the Iberian Pyritic belt using an array of geochemical and complementary molecular ecology techniques. Using an antibody microarray, we detected polymeric biomarkers (lipoteichoic acids and peptidoglycan) from Gram-positive bacteria throughout the borehole. DNA microarray hybridization confirmed the presence of members of methane oxidizers, sulfate-reducers, metal and sulfur oxidizers, and methanogenic Euryarchaeota. DNA sequences from denitrifying and hydrogenotrophic bacteria were also identified. FISH hybridization revealed live bacterial clusters associated with microniches on mineral surfaces. These results, together with measures of the geochemical parameters in the borehole, allowed us to create a preliminary scheme of the biogeochemical processes that could be operating in the deep subsurface of the Iberian Pyrite Belt, including microbial metabolisms such as sulfate reduction, methanogenesis and anaerobic methane oxidation.

Bioremediation of oily hypersaline soil and water via potassium and magnesium amendment.

Ten hydrocarbonoclastic halobacterial species and 5 haloarchaeal species that had been isolated on a mineral medium with oil as the sole carbon source grew better and consumed more crude oil, as measured by gas-liquid chromatography, in media receiving between 0.50 and 0.75 mol/L KCl and between 1.50 and 2.25 mol/L MgSO4. Chemical analysis revealed that within a certain limit, the higher the KCl and MgSO4 concentrations in the medium, the more K⁺ and Mg²âº, respectively, was accumulated by cells of all the tested halobacteria and haloarchaea. Also, in experiments in which total natural microbial consortia in hypersaline soil and water samples were directly used as inocula, the consumption of hydrocarbons was enhanced in the presence of the above given concentrations of KCl and MgSO4. It was concluded that amendment with calculated concentrations of K⁺ and Mg²âº could be a promising practice for hydrocarbon bioremediation in hypersaline environments.

Production of biosurfactant on crude date syrup under saline conditions by entrapped cells of Natrialba sp. strain E21, an extremely halophilic bacterium isolated from a solar saltern (Ain Salah, Algeria).

A bacterial strain E21 was isolated from a sample of water collected in the salt lake located close to Ain Salah, Algeria. The analysis of 16S rRNA gene sequence had indicated that the strain had 93 % sequence similarity with the genus Natrialba sp. strain E21 (GenBank, FR750525.1) and was considered extremely halophilic. Production of biosurfactant by the strain E21 with free and entrapped cells was investigated using soluble starch in the saline conditions. Biosurfactant synthesis was followed by measuring the surface tension and emulsifying index 9 days under optimal conditions (40 °C, pH 7). Some diffusional limitations in alginate and agar beads affected the kinetics of biosurfactant production when compared to that obtained with free cells culture. The minimum values of surface tension were 27 and 30 mN m(-1) achieved after 9 days with free and immobilized cells, respectively, while the corresponding maximum E24 values were 65.3 and 62.3 %, respectively. The re-use of bacterial cells along with the limited cell losses provided by the immobilized system might lead to significant reduction of the biosurfactant production cost.

Plant components with specific activities against rumen methanogens.

A wide range of plant bioactive components (phytochemicals) have been identified as having potential to modulate the processes of fermentation in the rumen. The use of plants or plant extracts as natural feed additives has become a subject of interest not only among nutritionists but also other scientists. Although a large number of phytochemicals (e.g. saponins, tannins and essential oils) have recently been investigated for their methane reduction potential, there have not yet been major breakthroughs that could be applied in practice. A key tenet of this paper is the need for studies on the influence of plant components on methane production to be performed with standardized samples. Where there are consistent effects, the literature suggests that saponins mitigate methanogenesis mainly by reducing the number of protozoa, condensed tannins both by reducing the number of protozoa and by a direct toxic effect on methanogens, whereas essential oils act mostly by a direct toxic effect on methanogens. However, because the rumen is a complex ecosystem, analysis of the influence of plant components on the populations of methanogens should take into account not only the total population of methanogens but also individual orders or species. Although a number of plants and plant extracts have shown potential in studies in vitro, these effects must be confirmed in vivo.